Gliederung

Primary products of cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolism, such as 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs), contribute to the regulation of vascular tone and renal function. Secondary metabolites include hydroxy-EETs (HEETs), which act as high affinity PPARa ligands. In the present study, we showed that several recombinant CYP2C enzymes do not only function as AA but also as 20-HETE epoxygenases and that this capacity provides a novel pathway of HEET formation in the rat kidney. Renal microsomes were isolated from 7-week old male Sprague-Dawley (SD) and from fenofibrate-treated (30mg/kg/d for 3 weeks) SD rats. HEETs produced from radiolabeled AA, EETs and 20-HETE were analyzed by reverse-phase HPLC followed by normal-phase HPLC to resolve the regioisomers. Renal microsomes efficiently produced HEETs from both EETs and 20-HETE. Fenofibrate induced 20-HETE epoxygenase activity 3-fold and 11,12-EET hydroxylase activity about 2-fold. An antibody against CYP2C23 abolished conversion of 20-HETE to HEETs, indicating that CYP2C23 is the predominant 20-HETE epoxygenase in rat renal microsomes. Recombinant CYP2C23 and renal microsomes produced identical patterns of HEET regioisomers from 20-HETE (20,8,9-HEET and 20,14,15-HEET in a ratio of 9:1). 20,8,9-HEET was also the major secondary metabolite produced from AA by renal microsomes. Immunohistochemical analysis revealed a co-localization of CYP2C23 and CYP4A proteins in tubules of the outer medulla and in medullary rays. These results demonstrate the existence of two HEET-generating pathways: (1) CYP4A-dependent hydroxylation of AA followed by CYP2C23-dependent 20-HETE epoxygenation and (2) CYP2C23-dependent epoxygenation of AA followed by CYP4A-dependent EET hydroxylation. Given the ability of HEETs to activate PPARa, HEETs may be important for the regulation of lipid metabolism and control of inflammation in the kidney.